(2015) 1-1 : 13-16
Research Article
13
Ekin
Journal of Crop Breeding and Genetics
(2015) 1-1:13-16
www.ekinjournal.com
Analysis of Ukrainian Polissya and Forest-steppe winter wheat (Triticum aestivum
L.) cultivars for the presence of “resistant” allelic state of non-race-specific disease
resistance locus Lr34/Yr18/Pm38
Ievgenij V. Zaika1 Anatolii V. Karelov2,3
Vasyl M. Starychenko1
Natalija O. Kozub2
Igor O. Sozinov2
Oleksij O. Sozinov2,3 National Science Center ”Institute of Agriculture of NAAS Ukraine”08162, Kiev oblast, Ukraine
Institute of Plant Protection of NAAS Ukraine, 03022, Kiev, Ukraine
3
Institute of Food Biotechnology and Genomics NAS of Ukraine, 04123, Kiev, Ukraine
Corresponding author: e-mail: [email protected]
1
2
Citation:
Zaika IV, Karelov AV, Kozub NO, Sozinov IO, Sozinov OO and Starychenko VM 2015. Analysis of Ukrainian Polissya and Forest-steppe winter
wheat (Triticum aestivum L.) cultivars for the presence of “resistant” allelic state of non-race-specific disease resistance locus Lr34/Yr18/Pm38. Ekin J
Crop Breed and Gen 1-1:13-16.
Received: 12.06.2014
Accepted: 27.09.2014
Published Online: 15.01.2015
Printed: 25.01.2015
ABSTRACT
Resistance against rusts and powdery mildew is one of the most important traits of modern wheat cultivars, because in
some years these diseases can significantly reduce crop yields. The genes that confer durable non-race-specific resistance
are of interest to many breeding programs. One of them is Lr34/Yr18/Pm38. This multiple resistance locus is associated
with partial and durable resistance to rust diseases, powdery mildew and tolerance to barley yellow dwarf virus. We used
marker caISBP1 (insertion site-based polymorphism marker) specific for the resistance, associated with the Lr34/Yr18/
Pm38 gene. It is a co-dominant marker between loci coding ABC-transporter and cytochrome P450 in the region involved
in resistance expression. We investigated a collection of 28 winter wheat cultivars of NSC ‘’Institute of Agriculture
NAAS Ukraine’’ developed in different periods. The “resistant” allelic state of the marker caISBP1 was identified in 11
cultivars (39% of the total number). The “susceptible” allelic state (the Lr34- allele) was identified in 9 wheat cultivars
(32%) and 8 (29%) cultivars were heterozygous. Our investigation confirmed the presence of the Lr34+ allele in Polissya
and Forest-Steppe winter wheat cultivars.
Keywords: rust diseases, adult plant resistance, winter wheat
Introduction
A search for cultivars can be donors of
resistance genes is a time-consuming but important
task of wheat breeding. For effective selection, a
breeder should have more or less solid data about
the resistance genes and the opportunity for their
quick identification during the breeding process.
Determining of resistance associated with some
genes by the phenotype may be inexact and timeconsuming. Detection of resistance gene accelerates
with the use of molecular DNA markers which can
rapidly identify allelic state of a gene in laboratory
with high precision. Using DNA markers in research
of resistance gene reduces the time and increases
efficiency and accuracy of the investigation in the
most cases. The rust diseases of wheat are caused
by fungi of the order Uredinales. There are three
species of wheat rusts: leaf rust (Puccinia triticina
f. sp. tritici), stem rust (P.graminis f. sp. tritici) and
yellow rust (P. striiformis f. sp. tritici). In some
years yield losses due leaf rust could reach 15-20%,
stem rust – 60-70%, yellow rust – 20% and more
(Peresypkin et al. 1990; Bublyk et al. 1999).
Rust fungi suppress the process of synthesis of
HMW glutenin components in kernels which affects
bread-making properties. Also they decline the rate
of synthesis and deposition of starch and proteins in
endosperm causing puny grain (Kolyuchyy et al. 2007).
Breeders can use wide non-race-specific adult
plant resistance (APR) genes which stay effective
for a long period. APR is associated with reduction
of disease development, slowdown of pathogen
haustorium penetration in cells of host plant
(Krattinger et al. 2009).
© Plant Breeders Union of Turkey (BISAB)
14
Lr34 is one of the most important APRs genes
in wheat breeding due to its wide spectrum resistance
and stability. It is present in about 50% of cultivars
worldwide and was a component of resistance of
highly productive cultivars at the start of green
revolution (Hoisington et al. 1999). The Lr34 gene
has been staying effective against rusts more than 100
years (Krattinger et al. 2009; Dakouri et al. 2010).
This multiple resistance locus is associated with
partial and durable resistance to rust diseases (Dyck et
al. 1987; McIntosh et al. 1992; Singh 1992; Kolmer et
al. 2011), powdery mildew (Blumeria graminis (DC.)
Speer) and tolerance to barley yellow dwarf virus
(Spielmeyer et al. 2005; Singh 1993). The gene was
localized at the short arm of the 7D chromosome of
wheat (Dyck et al. 1987). The Lr34 locus putatively
codes the ATP-binding cassette (ABC)-transporter,
a protein localized at the cytoplasmic membrane
(Krattinger et al. 2009). A number of molecular marker
linked to this gene have been developed (Bossolini
et al. 2006, Lagudah et al. 2009). Among them there
are the markers localized in the Lr34 locus or the
regulated ones (Dakouri et al. 2010). We used the
marker caISBP1 (insertion site-based polymorphism
marker) specific for the resistance, associated with
the Lr34/Yr18/Pm38 gene. It is a co-dominant marker
between the ABC-transporter and cytochrome P450
in region involved in resistance expression (Dakouri
et al. 2010).
The aim of our investigation was detecting
the allelic state of the Lr34/Yr18/Pm38 locus using
DNA markers because cultivars of NSC “Institute
of Agriculture NAAS” have never been investigated
for the presence of this resistance. The resistanceassociated allele of the Lr34/Yr18/Pm38 gene can
be involved in breeding process to obtain cultivars
with resistance to multiple pathogens in Polissya
and Forest-Steppe due the positive influence of its
climatic conditions on Lr34 resistance expression.
Materials and methods
The DNA was extracted from kernels of 28
winter wheat cultivars developed in periods from
1971 to 1990 and 1992 – 2012 in Cereal Breeding and
Seed Growing Department of the National Science
Centre “Institute of Agriculture NAAS Ukraine”:
Polesskaya-90, Polesskaya-95, Kolectivnaya-77,
Shchedraya-Polesya, Polesskaya-71, Polesskaya-70,
Polesskaya-80, Polesskaya-87, Polesskaya-107,
Polesskaya-bezostaya, Kievskaya-73, Polesskaya-29,
Polesskaya-1259,
Polesskaya-92,
Miryutinka,
Zhuravka, Stolichna, Epilog, Gnom, Krayevyd,
Artemida, Kopilivchanka, Analog, Benefis. The
cultivars Olgana, IZ49-12 (Kesaria-Poliska) and
IZ15-12 (Pamyati-Girka) are under field trials. The
cultivar Kievskaya-polukarlikovaya was developed
in cooperation with the Institute of Plant Physiology
and Genetics of NAS (Kiev, Ukraine). Seeds were
obtained from the collection of NSC “Institute of
Agriculture NAAS” and the National Centre of
Plant Genetic Resources (Kharkov, Ukraine). These
cultivars were released for growing condition of
Forest-Steppe and Polissya agroclimatic zones.
DNA was extracted using Diatom™ DNA
Prep 100 (NEOGENE®, Ukraine) kit by the standard
protocol from the bulk of 7 kernels. We used allelespecific marker caISBP1 (insertion site based
polymorphism marker) for the identification of the
allelic state of the Lr34/Yr18/Pm38 locus (Dakouri et
al. 2010). This codominant marker is located between
the Lr34 locus which encodes the ABC-transporter
and first cytochrome P450 in the site effecting the
resistance expression. The GenPak® PCR Core
(NEOGENE®, Ukraine) was used for PCR. The
results were visualized by electrophoresis in 2-2,5%
agarose gel with 1xTBE buffer and stained with
ethidium bromide. The amplified DNA fragments
of 509 b.p. in length were obtained in case of
“resistance” allelic state of the marker caISBP1 (the
Lr34+ allele). In case of the “susceptible” allelic state
of the marker (the Lr34- allele) fragments of 391 b.p.
in length were observed (Dakouri et al. 2010). The
cultivars Chinese Spring (the Lr34+ allele, St+) and
Renan (the Lr34- allele, St-) were used as standards.
Marker of molecular mass was GeneRulerTM 50 bp
DNA Ladder ready-to-use (Fermentas, Lithuania).
Results and discussion
Among 28 cultivars developed in the Institute of
Agriculture 11 showed the Lr34+ allele of the marker
caISBP1. They are Stolichna, Artemida, Analog,
Benefis, Polesskaya- 70, Polesskaya-bezostaya,
Kievskaya-73, Kievskaya - polukarlikovaya,
Polesskaya - 80, Zhuravka, IZ15-12, (Table 1). On
the Figure 1 represented the electropforegrams of
several cultivars showed different results.
In 9 cultivars we detected the “susceptible”
allelic state of the marker caISBP1: Polesskaya-90,
Kolectivnaya-77,
Shchedraya-Polesya,
Epilog,
Gnom, Krayevyd, Kopilivchanka, Olgana and
Polesskaya-1259. Heterozygosity was detected
in 8 samples: Polesskaya-95, Polesskaya-71,
Polesskaya-87, Polesskaya-107, Polesskaya-29,
Polesskaya-92, Miryutinka, IZ49-12. Resistance
associated with Lr34 is common in cultivars developed
in period before 1990 and 1992-2012 equally.
Our results are in agreement with the previous
publication of Karelov et al. (2011) showing that the
(2015) 1-1 : 13-16
15
Lr34+ allele was widely spread among Ukrainian alleles that possibly derived from Bezostaya1, which
bread wheat cultivars. A sufficiently high resistance could be selected due to high bread-making quality
conferred by Lr34 in cultivars adapted for the (Zaika et al. 2012, Labuschagne et al. 2002).
Thus we performed the investigation of 28 winter
Polissya and Forest-Steppe gives evidence of its
broad adaptive value, which does not lose the value wheat cultivars of NSC ‘’Institute of Agriculture
nowadays. The specificity of breeding process in the NAAS Ukraine’’ developed in different periods. For
Institute of Agriculture (using moderate infectious molecular diagnostic we used the loci-specific marker
backgrounds) may account for selection of breeding caSNBP1 of the Lr34/Yr18/Pm38 gene conferring
material with the Lr34+ allele. It is known, that APR against several phytopathogens. Presence of
the resistance-associated
allele
the“Institute
marker was
main genetic
sourcestate
of Lr34
resistance was cultivar
TABLE
1 Allelic
of Lr34/Yr18/Pm38
loci in winter
bread wheat cultivars
of of
NSC
Bezostaya1, used as a parental component by identified in 11 cultivars (39% of the total number).
breeders.
This cultivar
has a unique combination These cultivars can be used in breeding programs as
of Agriculture
NAAS”
of adaptability, disease resistance and high bread donors of the resistance conferred by the Lr34/Yr18/
making quality (Morgounov et al. 2011). Analysis Pm38 gene.
of seed storage proteins loci ofYear
investigated
cultivars
of Allelic
Year of Allelic
Cultivar
Cultivar
showed the
presence of gliadin
and
HMW
glutenin
release
state
release
state
1971
+/-in winterPolesskaya-95
+/Table Polesskaya-71
1 Allelic state of Lr34/Yr18/Pm38
loci
bread wheat cultivars1996
of NSC “Institute
of Agriculture
NAAS”.
Polesskaya-70
1974
+
Kopilivchanka
2003
Kievskaya-73
1974Year of + Allelic Stolichna
2005
+Year of Allelic
Cultivar
Cultivar
Kolectivnaya-77
1974release - state
Gnom
2007
- release
state
KievskayaPolesskaya-71
Polesskaya-95
+/1977 1971 + +/Artemida
2008
+ 1996
polukarlikovaya
Polesskaya-70
1974
+
Kopilivchanka
2003
Miryutinka
1978 1974 +/- +
Analog
2008
+ 2005
Kievskaya-73
Stolichna
+
Kolectivnaya-77
Gnom
Polesskaya-bezostaya
1981 1974 +
Benefis
2008
+ 2007
Kievskaya-Polukarlikovaya
+
Artemida
+
Polesskaya-80
1982 1977 +
Epilog
2009
- 2008
Miryutinka
1978
+/Analog
2008
+
Shchedraya-Polesya
1986
Krayevyd
2012
Polesskaya-Bezostaya
1981
+
Benefis
2008
+
Polesskaya-87
1990
+/Olgana
Polesskaya-80
1982
+
Epilog
2009
Polesskaya-92
1992 1986 +/- Zhuravka
+ 2012
Shchedraya-Polesya
Krayevyd
Polesskaya-87
Olgana
Polesskaya-90
1994 1990 - +/- Polesskaya-107
+/Polesskaya-92
1992
+/Zhuravka
+
Polesskaya-1259
1995
IZ15-12
+
Polesskaya-90
1994
Polesskaya-107
+/Polesskaya 29
1996
+/IZ49-12
+/Polesskaya-1259
1995
IZ15-12
+
«+»
–
“resistant”
allelic
state,
«-»
–
“susceptible”
allelic
state,
«+/-»
–
heterozygous
for
the
Polesskaya-29
1996
+/IZ49-12
+/Lr34+
allele) with allelic
the marker
caISBP1
«+» – “resistant”
state, «-»
– “susceptible” allelic state, «+/-» – heterozygous for the
Lr34+ allele) with the marker caISBP1
509 b.p.
391 b.p.
L
1
2
3
4
5
6
7
8
9
10
11 12 13 L
Figure 1 Electrophoregram of PCR products, obtained with DNA samples of bread winter wheat
FIGURE
1 that
Electrophoregram
of PCR 1–
products,
obtained2with
DNA samples
bread winter
and primers
flank marker caISBP1:
Polesskaya-80;
– Polesskaya-87;
3 –ofPolesskaya-107;
4 –wheat
Zhuravka;
5 – Stolichna;
6 – Epilog;
– Gnom; 81–– Krayevyd;
9 – Artemida;
10 –
and primers
that flank
marker7caISBP1:
Polesskaya-80;
2 – Polesskaya-87;
3 –
Kopilivchanka; 11 – Analog; 12 – Renan (St-); 13 – Chinese Spring (St+); L – marker of molecular
Polesskaya-107;
– Zhuravka; 5 – Stolichna; 6 – Epilog; 7 – Gnom; 8 – Krayevyd; 9 –
weight
(50 bp DNA 4Ladder)
Artemida; 10 – Kopilivchanka; 11 – Analog; 12 – Renan (St-); 13 – Chinese Spring (St+); L –
marker of molecular weight (50 bp DNA Ladder)
© Plant Breeders Union of Turkey (BISAB)
16
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